Piloty's acid derivative with improved nitroxyl-releasing characteristics

Article abstract of DOI:10.1016/j.bmcl.2013.02.062

Recent studies have shown that nitroxyl (HNO) (¹HNO/ ³NO^-), which is the one-electron-reduced form of nitric oxide (NO), has unique biological activities, especially in the cardiovascular system, and HNO-releasing agents may have therapeutic potential. Since few HNO donors are available for use under physiological conditions, we synthesized and evaluated a series of Piloty's acid (PA) derivatives and evaluated their HNO-releasing activity under physiological conditions. N-Hydroxy-2- nitrobenzenesulfonamide (17) was the most efficient HNO donor among our synthesized PA derivatives, including the lead compound, 2-bromo-N- hydroxybenzenesulfonamide (2). The high HNO-releasing activity is suggested to be due to electronic and steric effects. Compound 17 may be a useful tool for biological experiments.

Full text of DOI:10.1016/j.bmcl.2013.02.062

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2340–2343
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Piloty’s acid derivative with improved nitroxyl-releasing
characteristics
a
a
a
Kazuyuki Aizawa a, Hidehiko Nakagawa a,b, , Kazuya Matsuo , Kodai Kawai , Naoya Ieda ,
⇑
Takayoshi Suzuki a, , Naoki Miyata a,
⇑
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
b Japan Science and Technology Agency, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Recent studies have shown that nitroxyl (HNO) (1HNO/3NOÀ), which is the one-electron-reduced form of
nitric oxide (NO), has unique biological activities, especially in the cardiovascular system, and HNO-
releasing agents may have therapeutic potential. Since few HNO donors are available for use under phys-
iological conditions, we synthesized and evaluated a series of Piloty’s acid (PA) derivatives and evaluated
their HNO-releasing activity under physiological conditions. N-Hydroxy-2-nitrobenzenesulfonamide (17)
was the most efficient HNO donor among our synthesized PA derivatives, including the lead compound,
2-bromo-N-hydroxybenzenesulfonamide (2). The high HNO-releasing activity is suggested to be due to
electronic and steric effects. Compound 17 may be a useful tool for biological experiments.
Ó 2013 Elsevier Ltd. All rights reserved.
Article history:
Received 13 December 2012
Revised 6 February 2013
Accepted 13 February 2013
Available online 26 February 2013
Keywords:
Nitroxyl
HNO
Nitric oxide
Piloty’s acid
GC–MS
Nitroxyl (HNO) (1HNO/3NOÀ) is the one-electron-reduced form
of nitric oxide (NO).1 It has significant pharmacological effects that
differ, at least in mechanism, from those of NO, including enzyme
inhibition (ALDH,2 GAPDH,3) positive inotropy,4 vasodilation,5 and
cardioprotection.6 Therefore, HNO might have potential therapeu-
tic applications, for example, in alcohol dependency, heart failure
or breast cancer. It also reacts with thiols7 and phosphine,8 and
nitrosylates Fe, Mn and Co,9 so that it is likely to have other inter-
esting biological effects as well. However, HNO dimerizes sponta-
neously to form nitrous oxide (N2O),10 so it is hard to store.
Therefore, it is practically indispensable to use HNO donors
(HNO-releasing compounds) in biological experiments. Various
HNO donors have so far been developed2a,5,11 (Fig. 1).
N-Hydroxybenzenesulfonamide 1, or Piloty’s acid (PA) (Fig. 2),
is an HNO donor first reported in 1896.11b However, it is less
widely used in biological experiments than Angeli’s salt (AS), since
PA decomposes and releases HNO under basic conditions (for
example, the half-life of PA in aqueous solution is 561, 90, and
33 min at pH 8.0, 9.0, and 10.0, respectively12). In 2011, Toscano
et al. reported that 2 (Fig. 2) was a HNO-selective donor and re-
leased HNO at physiological pH.13 In the light of their results, we
hypothesized that other aryl-substituted PAs might also be effec-
tive HNO-releasing compounds. Here, we report the synthesis of
various PA derivatives that are decomposed with release of HNO
in aqueous solution at physiological pH. Their HNO-releasing activ-
ities were evaluated by GC–MS analysis. Among these compounds,
17 has the highest HNO-releasing activity so far reported for a PA
derivative.
Although many HNO donors have been reported, a few of them
have been be often used for biological experiments.
However, it would be generally desirable to investigate with
multiple donors, to ensure that the results are actually due to
HNO, but not to the donor itself or its degradation products. There-
fore, it is important to develop new HNO donors that release HNO
selectively at physiological pH.
N-Hydroxyarylsulfonamides were synthesized according to
Bois’s method.14 Briefly, commercially available arylsulfonyl chlo-
rides were condensed with hydroxylamine under basic conditions
to give corresponding N-hydroxyarylsulfonamide derivatives. The
aryl-substituted PA derivatives 2–23 were obtained in good to
moderate yield (Scheme 1).
We evaluated the HNO-releasing activity of the synthesized PA
derivatives by GC–MS detection of nitrous oxide (N2O), the dimer-
ization product of HNO. Since dimerization of HNO follows a sec-
ond-order rate equation, we prepared a standard curve of N2O as
a function of AS concentration, and evaluated the N2O formation
from the PA derivatives as the AS equivalent amount. We found
⇑
Corresponding authors. Tel./fax: +81 52 836 3408 (H.N.); tel./fax:+81 52 836
3407 (N.M.).
E-mail addresses: deco@phar.nagoya-cu.ac.jp (H. Nakagawa), miyata-n@phar.-
nagoya-cu.ac.jp (N. Miyata).
Present address: Kyoto Prefectural University of Medicine, 13 Taishogun,
Nishitakatsukasa-cho, Kita-ku, Kyoto 403-8334, Japan.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.062

K. Aizawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2340–2343
2341
-O
O-
O O
S
O-
N+
R=O-Na+ : IPA/NO
OCH2OAc : AcOM-IPA-NO
OCH=CH2 : V-IPA-NO
OCH2CH2Ms
N N+
iPr
OH
H2N
N
C
R
Ph
N
O-
2Na+
Angeli's salt
N
H
N
H
Piloty's acid
Cyanamide
O2N
O
N
R
N
O
O
O
N
H
R= CH3, tBu, CF3
R
O
O
O
X=CH2, O
R
N
X
R
Acyl nitroso
compounds
Acyloxy nitroso
compounds
R= Me, CH2CH2COOH
Figure 1. Currently available HNO donors.
O
O
HNO
O O
O O
S
S
OH
O
S
N
S
O
O
Base
H
Ar
N
H
H
Ar
N
Ar
O
H
X
N-hydroxyaryl-
arylsulfinic anion
X= H: 1, Piloty's acid (PA)
X= Br: 2, 2-bromo Piloty's acid (2BrPA)
sulfonamide
Scheme 2. Proposed mechanism of HNO release from Piloty’s acid derivatives.
Figure 2. PA and 2BrPA.
the peak of N2O disappeared, showing that it was dependent on
HNO production (data not shown). We also estimated the half-life
(t1/2) of PA derivatives. That of 17 is very fast (2.10 0.48 min)
while that of 1 and 2 is relatively slow (see Supplementary data,
Table S1). Therefore, it is suggested that N2O production shown
in Figure 3 is derived from dimerization of HNO released by
decomposition of PA derivatives.
As shown in Figure 3, PA derivatives bearing an electron-with-
drawing group (EWG) at the ortho-position were good HNO-
releasers (2, 6, 9, 11, 14, and 17). Compound 17 released the great-
est amount of HNO among our synthesized PA derivatives. This re-
sult can be explained as follows.
First, it was reported that the reaction of HNO release from PA
starts with deprotonation of the hydroxyl group16 (Scheme 2).
Therefore, EWGs such as –NO2, –CF3, –OCF3 would decrease the
pKa and promote deprotonation of the hydroxyl group. However,
mesityl (2,4,6-trimethylphenyl) and trisyl (2,4,6-triisopropyl-phe-
nyl) groups, regardless of their electron-donating property, en-
hanced HNO release, whereas meta- or para-derivatives had little
or no effect (Fig. 3), though ortho-substitution with other EWGs
was effective. These results can not be explained by electric effect
and we should discuss other effect. Second, SAR study in Figure 3
showed that bulkiness at the o-position with respect to the N-
hydroxysulfonamide group has a critical effect on HNO-releasing
activity and are consistent with a previous report from Doctorovich
and co-workers.17 We considered that bulky substituents at both
O
O
O O
NH2OH•HCl
DMAP
OH
S
S
Cl
N
H
pyridine
R
R
ice-cooling to r.t.
substituted benzene-
sulfonyl chloride
Ar-substituted
Piloty's acid
2
3
4
5
6
10
11
12
13
14
17
18
19
20
21
: R = 2-Br (48%)
: R = 3-Br (38%)
: R = 4-Br (36%)
: R = 2-F (79%)
: R = 2-Cl (78%)
7 : R = 2-Me (32%)
8 : R = 2-OMe (37%)
9 : R = 2-COOMe (48%)
: R = 2-CN (32%)
: R = 2-CF3 (83%)
: R = 3-CF3 (85%)
: R = 4-CF3 (57%)
: R = 2-OCF3 (92%)
: R = 2-NO2 (79%)
: R = 3-NO2 (97%)
: R = 4-NO2 (91%)
: R = 4-NHAc (20%)
i
: R = 2,4,6- Pr (69%)
15 : R = 3-OCF3 (88%) 22 : R = 2,4,6-Me (74%)
16 : R = 4-OCF3 (82%) 23 : R = 2,3,4,5,6-Me (38%)
Scheme 1. Synthesis of PA derivatives 2–23.
that 17 released the greatest amount of N2O under our experimen-
tal conditions (Fig. 3). Indeed, the release was 3 times that from
equimolar AS, a widely used HNO donor, and 1.5 times that from
2. We considered that higher and faster decomposition of PA deriv-
atives to sulfinic acid derivatives resulted in more efficient HNO-
releasing from them (Scheme 2).
We confirmed that when 2-mercaptoethanol (2-ME), an HNO
scavenger,11g,15 was added to solutions of AS and PA derivatives,
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0min.
10min.
20min.
30min.
Figure 3. N2O formation from PA derivatives. AS (500
lM in 50 mM Tris–HCl pH 7.5) or a PA derivative (500
lM in 50 mM Tris–HCl pH 7.5, 20% DMSO) was put in a cuvette at
room temperature and the reaction headspace gas (50
3 min) was calculated by the instrument software.
lL) was sampled and injected into the Rt-QPLOT column of a GC–MS instrument. The area of the N2O peak (tR = ca.

2342
K. Aizawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2340–2343
ortho positions would thermodynamically destabilize the molecule
and promote HNO release to yield thermodynamically stable ben-
zenesulfinic acid derivatives (Scheme 3). In other words, bulky
substituents reinforce HNO-releasing activity.
However, Figure 3 also showed that 23 had high HNO-releasing
activity than 22 though 3 and 5 position did not contribute the ste-
ric hinderance, and are EDGs. We carried out DFT calculation of 22
and 23 by Spartan ’08, to suggest that the energy gap of 23 be-
tween PA form and sulfinic acid form (decomposed product) is
smaller than that of 22 (see Supplementary data Table S2). There-
fore, 23 would release much HNO than 22.
A previous report showed HNO react NO to form N2O and ni-
trite.18 Since PA (1) could release NO under physiological condi-
tions,19 if PA derivatives could release HNO and NO at the same
time, N2O can be formed from the reaction of HNO and NO. Reac-
tion rate constants of N2O production from HNO dimerization and
from the reaction of HNO and NO are as follows.18
N2O-releasing activity is related not only to the electronic factor,
but also to a steric or solvent factor. The activity of previously re-
ported 2 is consistent with these considerations and recent report
supported the importance of electron-withdrawing/donating
groups for HNO-releasing.17
Finally, we identified the degradation product of 17. If 17
decomposes according to Scheme 2, 2-nitrobenzenesulfinic acid
would be formed. We synthesized an authentic sample of 2-nitro-
benzenesulfinic acid according to Kudo’s method20 and compared
the retention time (tR) of the degradation product from that of
the authentic sample. The two were identical (Fig. 5). This supports
the idea that 17 decomposed according to proposed mechanism
(Scheme 2) to form 2-nitrobenzenesulfinic acid.
In conclusion, we synthesized PA derivatives and found that 17
has the highest HNO-releasing activity among our synthesized
compounds, and is more active than 1 or 2. The electron-with-
drawing group at the ortho-position was suggested to facilitate
HNO release and appropriate bulkiness of the substituent at the
o-position would be important for HNO-releasing properties
(Fig. 6). Based on these results, we are now investigating the utility
of PA derivatives for biological applications.
k= 8.0 ×106 m-1s-1 (1)
k= 5.6 ×106 m-1s-1 (2)
k= 5.4 ×109 m-1s-1 (3)
k= 2.6 ×102 m-1s-1 (4)
HNO + HNO
HNO + •NO
N2O + H2O
N2O2
+ H
N2O2
+ •NO
N3O3
N3O3
N2O + NO2
100
O
O
80
60
40
20
0
S
OH
N
The rate-determining steps are Eq. 1 (HNO dimerization) and Eq. 4
(reaction of HNO and NO) and the reaction rate constant of the for-
mer is 104-fold faster than that of the latter. Therefore, the N2O pro-
duction from the latter process would be small enough to be
ignored, and it is suggested that N2O formation shown in Figure 3
would come from HNO dimerization.
In order to evaluate the above hypothesis, we examined the cor-
relation of pKa of corresponding substituted benzoic acid, a param-
eter that aims to evaluate the electronic effect of electron-
withdrawing/donating groups, with HNO release. We found that
N2O release from the synthesized PA derivatives was correlated
with the value of the pKa of corresponding substituted benzoic acid
(Fig. 4). If HNO release was related only to the electronic effect of
H
NO2
17
(authentic)
0
0
0
5
5
5
10
10
10
15
20
25
30
35
35
35
Retention time (min)
100
80
60
40
20
0
SO2H
NO2
sulfinic acid (authentic)
15
20
25 30
Retention time (min)
100
80
60
40
20
0
electron-withdrawing/donating groups, Figure
4 would have
shown a linear relationship. However, the scatter suggests that
decomposed 17
15
20
25
30
R
R
O
S
Retention time (min)
O O
S
HNO
OH
N
OH
Figure 5. HPLC detection of 2-nitrobenzenesulfinic acid, which is the degradation
product of 17. All samples were diluted with DMSO and 50 mM Tris–HCl pH 7.5
(final 500 lM, 20% DMSO), followed by incubation for 1 h at 37 °C.
H
R = Me or iPr
R
R
R
R
hindered
less hindered
Scheme 3. Proposed mechanism of promotion of HNO release from 21, 22 and 23.
Electron with drawing group
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
F
Cl
Br
iPr
CF3
OCF3
NO2
iPr
iPr
COOMe
2
2.5
3
3.5
K
a of substituted benzoic acid
4
4.5
sterically hindering group
p
Figure 6. Schematic illustration of factors affecting the HNO-releasing activity of
Figure 4. Correlation between N2O release and pKa of substituted benzoic acid.
PA derivatives.

K. Aizawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2340–2343
2343
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Acknowledgments
This work was supported in part by Grants-in-Aid for
Scientific Research on Innovative Areas (Research in a Proposed
Research Area) (No. 2117514 to H.N.) and Grands-in-Aid for
Scientific Research (No. 22590103 to H.N.) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan,
and JST, PRESTO.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.062.
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